| ObjectivesPogostemonis Herba belongs to homology of medicine and food.It is not only used as the main compositions in more than 40 kinds of Traditional Chinese Medicine?TCM?preparations on the market,such as Huoxiang Zhengqi Liquid and HuoDan pill,but also widely consumed in the form of beverages,candy,baked products,and common botanical ingredients in functional foods.It occupies a significant share of the market and has great potential for development.Therefore,it is important to control the quality of medicinal materials and volatile oil of Pogostemonis Herba.Pogostemonis Herba,an important medicine with the function of resolving turbidity with aroma,is commonly used in the clinical treatment of digestive diseases in TCM.Numerous studies focus on the gastrointestinal protection of its volatile ingredients.Patchouli alcohol,pogostone and?-patchoulene are the higher content of fragrant ingredients in Pogostemonis Herba.Patchouli alcohol and pogostone have been demonstrated as the material foundation of efficacy of Pogostemonis Herba in resolving turbidity with aroma,whetting appetite and arresting vomiting.They have protection against gastric ulcer and ulcerative colitis.Patchouli alcohol even is an indicator component of the quality of patchouli medicinal materials and volatile oil,as China's national and local standards of medicinal herbs.However,medicinal materials and volatile oils of Pogostemonis Herba are mixtures with many components.Only patchouli alcohol is used as an indicator component to control the quality of medicinal materials and essential oils is insufficient.There are many other indicator components can represent for the material foundation of efficacy of Pogostemonis Herba.It is necessary to combine patchouli alcohol with other indicator components to form the indicator components group,which can improve the quality standards and strictly control the quality of herbs and volatile oils on the market.Therefore,it is necessary to explore the ingredient that can be demonstrated as the material foundation of efficacy of Pogostemonis Herba.At present,studies of the protective effect of Pogostemonis Herba against gastrointestinal diseases mainly focus on its components,including patchouli alcohol and pogostone.However,there is a lack of research on other monomer components,such as?-patchoulene.Chemical structure of?-patchoulene is similar to patchouli alcohol.Previous study found that patchouli alcohol can be converted to?-patchoulene under acidic conditions.Gastric juice just is acidic solution,whether patchouli alcohol can be converted to?-patchoulene in gastric juice is still unknown.If?-patchoulene is a metabolite of patchouli alcohol in gastric juice,whether it has protective effect against gastric ulcer and ulcerative colitis,just like patchouli alcohol.Meanwhile,there is no research underlying the mechanism of protective effect of?-patchoulene against gastric ulcer and ulcerative colitis.Additionally,?-patchoulene is one of the aromatic ingredients in Pogostemonis Herba and has a high content.If?-patchoulene is comfirmed to have protective effect against gastric ulcer and ulcerative colitis,it undoubtedly can be the material foundation of efficacy of Pogostemonis Herba,which can provide scientific basis for Pogostemonis Herba to treat gastrointestinal disorders.Meanwhile,it can form the the indicator components group with patchouli alcohol and pogostone,which can improve quality of medicinal materials and volatile oil of Pogostemonis Herba and be conducive to the healthy development of Pogostemonis Herba medicine market.Therefore,it is very meaningful to investigate the protective effect of?-patchoulene against gastric ulcer and ulcerative colitis.This study intended to investigate the metabolism of patchouli alcohol in gastric juice and the mechanism of conversion of patchouli alcohol to?-patchoulene through metabolic test in vitro and in vivo.Then the protective effect of?-patchoulene and underlying mechanism against gastric ulcer would be investigated through ethanol-induced gastric epithelial GES-1 cells injury model,as well as ethanol and indomethacin-induced gastric ulcer in rats.Meanwhile,protective effect of?-patchoulene and underlying mechanism against ulcerative colitis would be investigated through lipopolysaccharide?LPS?-induced RAW264.7 macrophage inflammatory model and dextra sulfate sodium?DSS?-induced ulcerative colitis in mice.Methods1.Metabolic Study of patchouli alcohol in gastric juiceTo establish in vitro and in vivo model about the metabolic test in gastric juice.Metabolites of patchouli alcohol would be analysed using an Agilent Technologies GC7890A-MSD5975C system.Chromatographic separation was achieved with an HP-5MS5%Phenyl Methyl Silox column?30 m×250?m×0.25?m?.Helium?purity of 99.999%?was used as the carrier gas at a flow rate of 1.3 mL/min,and the injector split was 1:60.The injector temperature was 250?and the column oven temperature was programmed from50°C?held for 2 min?,increased to 120°C with a rate of 60?/min,and was then isothermal for 5 min.Subsequently,the temperature increased to 150?at 10?/min,followed by a 60?/min increase to 235?and was maintained at 235?for 15 min.Full scan data were obtained from 35 m/z to 400 m/z.2.Protective effect of?-patchoulene against gastric ulcerIn the model of ethanol-induced GES-1 gastric epithelial cells injury,the cell viability was detected by MTT assay after exposing to?-patchoulene.It intended to evaluate the protective effect of?-patchoulene on ethanol-induced injury in GES-1 gastric epithelial cells.Meanwhile,to evaluate the protective effect of?-patchoulene against ethanol and indomethacin-induced gastric ulcer through measuring ulcer areas and inhibition,observing histology of gastric mucosa with hematoxylin-eosin staining and scoring,as well as detecting the pH value of gastric juice.In order to elucidate the possible protective mechanism of?-patchoulene against ethanol-induced gastric ulcer,the contents of SOD,GSH,CAT and MDA were measured by assay kit,the levels of TNF-?,IL-1?and IL-6were measured by ELISA kit,the expressions of Fas,FasL and caspase protein were detected using immunohistochemistry,the gene expressions of c-fos,c-jun and miR-21were detected using qRT-PCR,and the phosphorylation levels of p65,I?B and ERK1/2were determined using Western blot.Additionally,In order to elucidate the possible protective mechanism of?-patchoulene against indomethacin-induced gastric ulcer,the contents of MPO were measured by assay kit,the levels of TNF-??E-selectin?P-selectin?MCP-1?ICAM-1?VCAM-1?VEGF and ET-1 were measured by ELISA kit,the expressions of COX-1 and COX-2 protein were detected using immunohistochemistry,the mRNA expressions of flt-1 and ETAR were detected using qRT-PCR,and the phosphorylation levels of I?B and JNK,as well as expression of PGE2 protein were determined using Western blot.3.Protective effect of?-patchoulene against ulcerative colitisIn the model of LPS-induced RAW264.7 macrophage inflammatory,the content of NO was detected by Griess reagent,the levels of PGE2,TNF-?,IL-6,IL-1?and IL-10 were measured by ELISA kits,as well as the mRNA expression of COX-2,iNOS,TNF-?,IL-6,IL-1?and IL-10 was detected using qRT-PCR after exposing to?-patchoulene.It intended to evaluate the anti-inflammatory activity of?-patchoulene on LPS-induced RAW264.7macrophage inflammatory.Meanwhile,to evaluate the protective effect of?-patchoulene against DSS-induced ulcerative colitis through recording the physical sign,measuring the colon length and spleen weight,observing histology of colonic mucosa with hematoxylin-eosin staining and scoring,as well as detecting the pH value of gastric juice.Additionally,in order to elucidate the possible protective mechanism of?-patchoulene against DSS-induced ulcerative colitis,the contents of MPO were measured by assay kit,the levels of TNF-?,ICAM-1,P-selectin and LBP were measured by ELISA kit,the apoptosis of intestinal mucosa was detected using TUNEL,the mRNA expressions of occluding and ZO-1 were detected using qRT-PCR.Meanwhile,the phosphorylation levels of I?B?and MLC2,as well as the expressions of ROCK1,TLR4,MyD88 and p65 proteins were determined using Western blot.Results1.Metabolic Study of patchouli alcohol in gastric juicePatchouli alcohol could be converted to?-patchoulene in hydrochloric acid,simulated gastric juice and rat gastric juice in vitro metabolic test.However,the conversion rate of patchouli alcohol to?-patchoulene in hydrochloric acid is better than that in simulated gastric juice and rat gastric juice.It is speculated that metabolic transformation of patchouli alcohol may be affected by various factors,such as pepsin,mucus,electrolytes and intrinsic factors.In vivo metabolic test,patchouli alcohol had been confirmed to transform to?-patchoulene,but the conversion is not high.The pH value of rat gastric juice was 1.5-5.5in vivo metabolic test while the pH value of rat gastric juice was 1.5-2.0 in vitro metabolic test,there is a difference between them.It is speculated that the pH value of gastric juice may be the important reason that affects metabolism of patchouli alcohol in gastric juice.The conversion of patchouli alcohol is significantly different in different pH conditions of simulated gastric juice.The smaller of pH value,the higher conversion of patchouli alcohol.And patchouli alcohol was not transformed when pH value of gastric juice is higher than3.5.2.Protective effect of?-patchoulene against gastric ulcerIn the model of ethanol-induced GES-1 gastric epithelial cells injury,the viability of GES-1 cells was significantly increased after exposing to different doses of?-patchoulene?10,20 and 40?mol/L?.After orally administration of different doses of?-patchoulene?10,20 and 40 mg/kg?,the ulcer areas were reduced,as well as histomorphology of gastric mucosa was improved with reduced score in the model of ethanol and indomethacin-induced gastric ulcer.In the same dosage,?-patchoulene is more effective than patchouli alcohol in preventing ethanol-induced gastric injury not the indomethacin-induced gastric ulcer.The pH value of gastric juice was about 2.5 in rats with ethanol-induced gastric ulcer,patchouli alcohol did not significantly transform in gastric juice after orally administration.However,the pH value of gastric juice was about 1.5 in rats with indomethacin-induced gastric ulcer,patchouli alcohol may all be transformed in gastric juice after orally administration.In the model of ethanol-induced gastric ulcer in rats,different doses of?-patchoulene could increase the contents of GSH,CAT,CAT and decrease the content of MDA to inhibit oxidative stress.Then?-patchoulene reduced the levels of TNF-?,IL-1?and IL-6,and down-regulate the phosphorylation levels of p65,I?B to inhibit the NF-?B signaling pathway and reduce the inflammatory response.?-patchoulene also could down-regulate the expressions of Fas,FasL and caspase-3proteins,up-regulate the gene expressions of c-fos,c-jun,miR-21,and the phosphorylation of ERK1/2 to inhibit gastric mucosal cell apoptosis.Additionally,in the model of indomethacin-induced gastric ulcer in rats,different doses of?-patchoulene could reduce the contents of MPO and the levels of TNF-??E-selectin?P-selectin?MCP-1?ICAM-1?VCAM-1,as well as down-regulate the phosphorylation of I?B?and JNK to inhibit the NF-?B and JNK signaling pathways and alleviate the inflammatory responses.?-patchoulene also up-regulate the expressions of COX-1,COX-2 and PGE2,promote the expression of VEGF and flt-1,and inhibit the expression of ET-1 and ETAR to promote angiogenesis and accelerate the healing of gastric ulcer.3.Protective effect of?-patchoulene against ulcerative colitisIn the model of LPS-induced RAW264.7 macrophage inflammatory,the contents of PGE2,NO,TNF-?,IL-6,IL-1?were decreased and IL-10 was increased while the mRNA expressions of COX-2,iNOS,TNF-?,IL-6,IL-1?were down-regulated and IL-10 was up-regulated after exposing to different doses of?-patchoulene?10,20 and 40?mol/L?.It is demonstrated that?-patchoulene could inhibit the inflammatory response in LPS-induced RAW264.7 macrophage.After orally administration of different doses of?-patchoulene?5,10 and 20 mg/kg?,the reduced body weights,diarrhea,shortened colon and DAI index were improved,as well as histomorphology of colonic mucosa was improved with reduced score in the model of DSS-induced ulcerative colitis.?-patchoulene is more effective than patchouli alcohol in preventing DSS-induced ulcerative colitis under the same dosage.The pH value of gastric juice was about 2.0 in rats with DSS-induced ulcerative colitis,most patchouli alcohol may be transformed in gastric juice after orally administration.In the model of DSS-induced ulcerative colitis in mice,different doses of?-patchoulene could reduce colonic mucosal apoptosis,decrease the content of LBP in tissues and serum,up-regulate the mRNA expression of occludin and ZO-1,downregulate the expression of ROCK1 protein and the phosphorylation of MLC2 to inhibit the ROCK-MLC signal pathway and protect the intestinal mucosal barrier function.Meanwhile,?-patchoulene could decrease the levels of TNF-?,ICAM-1,P-selectin and MPO,down-regulate the protein expression of TLR4 and MyD88,inhibit the phosphorylation of I?B?and the nuclear translocation of p65 subunit to inhibit TLR4-mediated NF-?B signal pathways and regulate immune function to reduce colonic mucosal injury.ConclusionPatchouli alcohol was confirmed to be transformed into?-patchoulene in gastric juice through in vitro and in vivo metabolic test.Its conversion rate may be related to pH value,pepsin,mucus,electrolytes and intrinsic factors of gastric juice,of which pH value was particularly important.?-patchoulene was confirmed to possess protective activity against ethanol-induced gastric ulcer and its effect was better than patchouli alcohol.The underlying mechanism of?-patchoulene against ethanol-induced gastric ulcer was related with its activity of anti-oxidation,anti-inflammatory and anti-apoptosis.Meanwhile,?-patchoulene was confirmed to possess protective activity against indomethacin-induced gastric ulcer and its effect was similar to patchouli alcohol.The underlying mechanism of?-patchoulene against indomethacin-induced gastric ulcer was related with its activity of anti-inflammatory and angiogenesis.At last,?-patchoulene was firstly confirmed to possess protective activity against DSS-induced ulcerative colitis and its effect was better than patchouli alcohol.The underlying mechanism of?-patchoulene against DSS-induced ulcerative colitis was related with its regulation of immune function and protection of intestinal mucosal barrier function.It was suggested that?-patchoulene possess protective effect against gastric ulcer and ulcerative colitis,as well as being one of the material foundation of efficacy of Pogostemonis Herba in treatment of digestive system diseases.This study also provided a theoretical basis to improve the quality standard of herbs and volatile oil of Pogostemonis Herba. |
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